EP2010648B1 - Delta-17-desaturases et leur utilisation dans la preparation d'acides gras polyinsatures - Google Patents

Delta-17-desaturases et leur utilisation dans la preparation d'acides gras polyinsatures Download PDF

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EP2010648B1
EP2010648B1 EP07775772A EP07775772A EP2010648B1 EP 2010648 B1 EP2010648 B1 EP 2010648B1 EP 07775772 A EP07775772 A EP 07775772A EP 07775772 A EP07775772 A EP 07775772A EP 2010648 B1 EP2010648 B1 EP 2010648B1
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desaturase
seq
nucleic acid
sequence
gene
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EP2010648A2 (fr
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Zhixiong Xue
Quinn Qun Zhu
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EIDP Inc
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EI Du Pont de Nemours and Co
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C12P7/6432—Eicosapentaenoic acids [EPA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0083—Miscellaneous (1.14.99)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0083—Miscellaneous (1.14.99)
    • C12N9/0087—Steroid 21-monooxygenase (1.14.99.10)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone

Definitions

  • This invention is in the field of biotechnology. More specifically, this invention pertains to the identification of nucleic acid fragments encoding ⁇ 17 fatty acid desaturase enzymes and the use of these desaturases in making long chain polyunsaturated fatty acids (PUFAs).
  • PUFAs long chain polyunsaturated fatty acids
  • PUFAs are important biological components of healthy cells and are recognized as: "essential" fatty acids that cannot be synthesized de novo in mammals and instead must be obtained either in the diet or derived by further desaturation and elongation of linoleic acid (LA;18:2 ⁇ -6) or ⁇ -linolenic acid (ALA; 18:3 ⁇ -3); constituents of plasma membranes of cells, where they may be found in such forms as phospholipids or triacylglycerols; necessary for proper development (particularly in the developing infant brain) and for tissue formation and repair; and, precursors to several biologically active eicosanoids of importance in mammals (e.g., prostacyclins, eicosanoids, leukotrienes, prostaglandins).
  • LA linoleic acid
  • ALA ⁇ -linolenic acid
  • a variety of different hosts including plants, algae, fungi and yeast are being investigated as means for commercial PUFA production.
  • Genetic engineering has demonstrated that the natural abilities of some hosts (even those natively limited to LA and ALA fatty acid production) can be substantially altered to result in high level production of arachidonic acid (ARA; 20:4 ⁇ -6), eicosapentaenoic acid (EPA; 20:5 ⁇ -3) and docosahexaenoic acid (DHA; 22:6 ⁇ -3).
  • ARA arachidonic acid
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • a ⁇ 15 desaturase is responsible for the conversion of LA to ALA
  • a ⁇ 17 desaturase is responsible for the conversion of ARA to EPA (although some ⁇ 17 desaturases can also use dihomo- ⁇ -linolenic acid (DGLA; 20:3 ⁇ -6) as a substrate to produce eicosatetraenoic acid (ETA; 20:4 ⁇ -3)).
  • DGLA dihomo- ⁇ -linolenic acid
  • ETA eicosatetraenoic acid
  • the isolated nucleic acid molecule of the invention is selected from the group consisting of an isolated nucleotide sequence encoding a ⁇ 17 desaturase enzyme as set forth in SEQ ID NO: 3 and an isolated nucleotide sequence that is completely complementary thereto.
  • the invention provides genetic constructs that are codon optimized for expression in Yarrowia sp. i.e. a chimeric gene comprising the isolated nucleic acid molecule of the invention operably linked to suitable regulatory sequences. Additionally the invention provides host Yarrowia cells transformed with the genetic constructs of the invention for expression and production of ⁇ -3 fatty acids.
  • Percent (%) PUFAs in the total lipid and oil fractions refers to the percent of PUFAs relative to the total fatty acids in those fractions.
  • PC phosphatidylcholine
  • PE phosphatidyletanolamine
  • TAG or oil triacylglycerol
  • PUFA biosynthetic pathway refers to a metabolic process that converts oleic acid to LA, EDA, GLA, DGLA, ARA, ALA, STA, ETrA, ETA, EPA, DPA and DHA. This process is well described in the literature (e.g., see PCT Publication No. W02006/052870 ). Briefly, this process involves elongation of the carbon chain through the addition of carbon atoms and desaturation of the molecule through the addition of double bonds, via a series of special desaturation and elongation enzymes (i.e., "PUFA biosynthetic pathway enzymes") present in the endoplasmic reticulim membrane.
  • PUFA biosynthetic pathway enzymes a series of special desaturation and elongation enzymes
  • Some desaturases have activity on two or more substrates. Based on this ability, these enzymes can be further classified with respect to their desaturase activities as being either "monofunctional” or "bifunctional". In some embodiments, it is most desirable to empirically determine the specificity of a fatty acid desaturase by transforming a suitable host with the gene for the fatty acid desaturase and determining its effect on the fatty acid profile of the host.
  • bifunctional ⁇ 17 desaturase refers to a ⁇ 17 desaturase that preferentially converts ARA to EPA and/or DGLA to ETA but additionally has limited ability to convert LA into ALA (thus exhibiting primarily ⁇ 17 desaturase activity and limited ⁇ 15 desaturase activity).
  • A17 desaturases can have specificities other than ⁇ 17 and ⁇ 15 desaturation that are not relevant in this classification. It should also be noted that the distinction between monofunctional and bifunctional ⁇ 17 desaturases is a practical one and not absolute; e.g., the same enzyme may function with either monofunctional or bifunctional ⁇ 17 desaturase activity, depending on the level of its expression or growth condition.
  • oleaginous microorganisms It is not uncommon for oleaginous microorganisms to accumulate in excess of about 25% of their dry cell weight as oil.
  • oleaginous yeast include, but are no means limited to, the following genera: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.
  • Stringency conditions can be adjusted to screen for moderately similar fragments (such as homologous sequences from distantly related organisms), to highly similar fragments (such as genes that duplicate functional enzymes from closely related organisms).
  • Post-hybridization washes determine stringency conditions.
  • One set of preferred conditions uses a series of washes starting with 6X SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2X SSC, 0.5% SDS at 45 °C for 30 min, and then repeated twice with 0.2X SSC, 0.5% SDS at 50 °C for 30 min.
  • a more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2X SSC, 0.5% SDS was increased to 60 °C.
  • Another preferred set of highly stringent conditions uses two final washes in 0.1 X SSC, 0.1 % SDS at 65 °C.
  • An additional set of stringent conditions include hybridization at 0.1X SSC, 0.1 % SDS, 65°C and washes with 2X SSC, 0.1 % SDS followed by 0.1X SSC, 0.1% SDS, for example.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.
  • the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T m for hybrids of nucleic acids having those sequences.
  • the relative stability (corresponding to higher T m ) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA.
  • a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least about 30 nucleotides.
  • the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.
  • gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques).
  • short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers.
  • a "substantial portion" of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence.
  • the instant specification teaches the complete amino acid and nucleotide sequence encoding particular Oomycete proteins.
  • the skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above, which encode a ⁇ 17 desaturase as set forth in SEQ ID NO: 4 and which are codon optimised for expression in Yarrowia.
  • the term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another.
  • adenosine is complementary to thymine
  • cytosine is complementary to guanine.
  • the instant invention also includes isolated nucleic acid fragments that are complementary to the complete sequences as reported in the accompanying Sequence Listing, as well as those substantially similar nucleic acid sequences as defined in the claims.
  • homology and “homologous” are used interchangeably herein. They refer to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. It is therefore understood, as those skilled in the art will appreciate, that the invention encompasses more than the specific exemplary sequences as defined in the claims.
  • homologous nucleic acid sequences are also defined by their ability to hybridize, under moderately stringent conditions (e.g., 0.5 X SSC, 0.1% SDS, 60°C) with the sequences exemplified herein, or to any portion of the nucleotide sequences disclosed herein and which are functionally equivalent to any of the nucleic acid sequences disclosed herein.
  • moderately stringent conditions e.g., 0.5 X SSC, 0.1% SDS, 60°C
  • Codon degeneracy refers to the nature in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide.
  • the skilled artisan is well aware of the "codon-bias" exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
  • “Chemically synthesized”, as related to a sequence of DNA, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well-established procedures or, automated chemical synthesis can be performed using one of a number of commercially available machines. "Synthetic genes" can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form gene segments that are then enzymatically assembled to construct the entire gene. Accordingly, the genes can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favoured by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell, where sequence information is available.
  • Gene refers to a nucleic acid fragment that expresses a specific protein, and that may refer to the coding region alone or may include regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.
  • Native gene refers to a gene as found in nature with its own regulatory sequences.
  • Chimeric gene refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.
  • Endogenous gene refers to a native gene in its natural location in the genome of an organism.
  • a “foreign” gene refers to a gene that is introduced into the host organism by gene transfer.
  • Foreign genes can comprise native genes inserted into a non-native organism, native genes introduced into a new location within the native host, or chimeric genes.
  • a “transgene” is a gene that has been introduced into the genome by a transformation procedure.
  • a “codon-optimized gene” is a gene having its frequency of codon usage designed to mimic the frequency of preferred codon usage of the host cell.
  • Coding sequence refers to a DNA sequence that codes for a specific amino acid sequence.
  • Suitable regulatory sequences refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures.
  • Promoter refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA.
  • a coding sequence is located 3' to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
  • 3' non-coding sequences and “transcription terminator” refer to DNA sequences located downstream of a coding sequence. This includes polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
  • the polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.
  • the 3' region can influence the transcription, RNA processing or stability, or translation of the associated coding sequence.
  • Antisense RNA refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene ( U.S. Patent No. 5,107,065 ; PCT Publication No. WO 99/28508 ).
  • the complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, or the coding sequence.
  • “Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNA that is not translated and yet has an effect on cellular processes.
  • operably linked refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).
  • Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
  • nucleic acid fragments that encode amino acid sequences that are at least 95% identical to the amino acid sequences reported herein. Indeed, any integer amino acid identity from 70% to 100% may be useful, such as 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
  • TAGs (the primary storage unit for fatty acids) are formed by a series of reactions that involve: 1.) the esterification of one molecule of acyl-CoA to glycerol-3-phosphate via an acyltransferase to produce lysophosphatidic acid; 2.) the esterification of a second molecule of acyl-CoA via an acyltransferase to yield 1,2-diacylglycerol phosphate (commonly identified as phosphatidic acid); 3.) removal of a phosphate by phosphatidic acid phosphatase to yield 1,2-diacylglycerol (DAG); and 4.) the addition of a third fatty acid by the action of an acyltransferase to form TAG.
  • a wide spectrum of fatty acids can be incorporated into TAGs, including saturated and unsaturated fatty acids and short-chain and long-chain fatty acids.
  • the " ⁇ 6 desaturase/ ⁇ 6 elongase pathway" can be utilized for formation of ⁇ -3 fatty acids as follows: (1) LA is converted to ALA, the first of the ⁇ -3 fatty acids, by a ⁇ 15 desaturase; (2) ALA is converted to STA by a ⁇ 6 desaturase; (3) STA is converted to ETA by a C 18/20 elongase; (4) ETA is converted to EPA by a ⁇ 5 desaturase; (5) EPA is converted to DPA by a C 20/22 elongase; and, (6) DPA is converted to DHA by a ⁇ 4 desaturase.
  • ⁇ -6 fatty acids may be converted to ⁇ -3 fatty acids; for example, ETA and EPA are produced from DGLA and ARA, respectively, by ⁇ 17 desaturase activity.
  • Alternate pathways for the biosynthesis of ⁇ -3/ ⁇ -6 fatty acids utilize a ⁇ 9 elongase and ⁇ 8 desaturase. More specifically, LA and ALA may be converted to EDA and ETrA, respectively, by a ⁇ 9 elongase; then, a ⁇ 8 desaturase converts EDA to DGLA and/or ETrA to ETA.
  • ⁇ -3/ ⁇ -6 fatty acids will depend on the host cell (and its native PUFA profile and/or desaturase/elongase profile), the availability of substrate, and the desired end product(s).
  • One skilled in the art will be able to identify various candidate genes encoding each of the enzymes desired for ⁇ -3/ ⁇ -6 fatty acid biosynthesis.
  • Useful desaturase and elongase sequences may be derived from any source, e.g., isolated from a natural source (from bacteria, algae, fungi, oomycetes, plants, animals, etc.), produced via a semi-synthetic route or synthesized de novo.
  • each particular desaturase and/or elongase it will also be useful to consider the conversion efficiency of each particular desaturase and/or elongase. More specifically, since each enzyme rarely functions with 100% efficiency to convert substrate to product, the final lipid profile of un-purified oils produced in a host cell will typically be a mixture of various PUFAs consisting of the desired ⁇ -3/ ⁇ -6 fatty acid, as well as various upstream intermediary PUFAs. Thus, consideration of each enzyme's conversion efficiency is also a variable to consider when optimizing biosynthesis of a desired fatty acid.
  • candidate genes having the appropriate desaturase and elongase activities can be identified according to publicly available literature (e.g., GenBank), the patent literature, and experimental analysis of organisms having the ability to produce PUFAs. These genes will be suitable for introduction into a specific host organism, to enable or enhance the organism's synthesis of PUFAs.
  • Preferred amino acid fragments are at least about 70%-80% identical to the PsD17S and PrD17S sequences herein, where those sequences that are at least about 85%-90% identical are particularly suitable and those sequences that are at least about 95% identical are most preferred.
  • Preferred PsD17S and PrD17S encoding nucleic acid sequences corresponding to the instant ORFs are those encoding active proteins and which are at least about 70%-80% identical to the nucleic acid sequences of PsD17S and PrD17S reported herein, respectively, where those sequences that are at least 85%-90% identical are particularly suitable and those sequences that are at least about 95% identical are most preferred.
  • Embodiments of the invention include nucleic acid molecules which are 95% identical to the nucleic acid sequence of PsD17S and methods of the invention use nucleic acid molecules which have 95% identity to PsD17 or PsD17S.
  • oilseed plant hosts can be transformed with the ⁇ 17 desaturases described herein.
  • Plants so transformed can be monocotyledonous plants or dicotyledonous plants, and preferably they belong to a class of plants identified as oleaginous (e.g., oilseed plants).
  • oilseed plant hosts include, but are not limited to: soybean ( Glycine and Soja sp. ), com ( Zea mays ), flax ( Linum sp .), rapeseed ( Brassica sp. ), primrose, canola, maize, safflower ( Carthamus sp. ) and sunflower ( Helianthus sp .).
  • Means for overexpression of fatty acid desaturases in oilseed plants are described in PCT Publication No. WO 2005/047479 .
  • Yarrowia lipolytica strain ATCC #20362 was purchased from the American Type Culture Collection (Rockville, MD). Y. lipolytica strains were usually grown at 28 °C on YPD agar (1% yeast extract, 2% bactopeptone, 2% glucose, 2% agar).
  • MM minimal medium
  • yeast nitrogen base DIFCO Laboratories, Detroit, MI
  • ammonium sulfate or amino acids 2% glucose, 0.1% proline, pH 6.1
  • Supplements of leucine, lysine and/or uracil were added as appropriate to a final concentration of 0.01% (thereby producing "MMLe”, “MMLys” and “MMU” selection media, each prepared with 20 g/L agar).
  • fatty acid analysis For fatty acid analysis, cells were collected by centrifugation and lipids were extracted as described in Bligh, E.G. & Dyer, W. J. (Can. J. Biochem. Physiol., 37:911-917 (1959 )). Fatty acid methyl esters were prepared by transesterification of the lipid extract with sodium methoxide ( Roughan, G., and Nishida I. Arch Biochem Biophys., 276(1):38-46 (1990 )) and subsequently analyzed with a Hewlett-Packard 6890 GC fitted with a 30-m X 0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard) column. The oven temperature was from 170°C (25 min hold) to 185°C at 3.5 °C/min.
  • PsD17 The sequence homology of PsD17 to all publicly available protein sequences contained in the "nr" database (see General Methods) was also determined by conducting protein-protein BLAST searches using PsD17 (SEQ ID NO:2) as the query sequence. Based on this analysis, PsD17 was found to share the most homology with the omega-3 fatty acid desaturase of Saprolegnia diclina (GenBank Accession No. AAR20444); specifically, PsD17 had 60% identity and 74% similarity with the amino acid sequence of GenBank Accession No. AAR20444 with an Expectation value of 7E-117.
  • the codon usage of the ⁇ 17 desaturase gene of Phytophthora sojae was optimized for expression in Yarrowia lipolytica, in a manner similar to that described in U.S. Patent 7,125,672 .
  • a codon-optimized ⁇ 17 desaturase gene (designated "PsD17S", SEQ ID NOs:3 and 4) was designed based on the coding sequence of PsD17 (SEQ ID NOs:1 and 2), according to the Yarrowia codon usage pattern ( PCT Publication No. WO 2004/101753 ), the consensus sequence around the 'ATG' translation initiation codon, and the general rules of RNA stability ( Guhaniyogi, G. and J.
  • PsD17S lacked the third and forth amino acid, as compared with the wild type PsD17; thus, the total length of PsD17S is 361 amino acids (SEQ ID NO:4).
  • the designed PsD17S gene was synthesized by GenScript Corporation (Piscataway, NJ) and cloned into pUC57 (GenBank Accession No. Y14837) to generate pPsD17S (SEQ ID NO:10; Figure 3A ).
  • pKUNF1-KEA (SEQ ID NO:11; Figure 3B ) comprises a chimeric FBAINm::E1 S::Pex20 gene.
  • the "FBAINm” promoter within this chimeric gene ( PCT Publication No. WO 05/049805 ; also identified as “Fba1 +intron” in Figure 3B ) refers to a synthetic promoter that is derived from the "FBAIN" promoter, wherein the FBAIN promoter refers to the 5' upstream untranslated region in front of the 'ATG' translation initiation codon of a fructose-bisphosphate aldolase enzyme (E.C.
  • the FBAINm promoter is modified from FBAIN, in that FBAINm has a 52 bp deletion between the ATG translation initiation codon and the intron of the FBAIN promoter (thereby including only 22 amino acids of the N-terminus) and a new translation consensus motif after the intron. Furthermore, while the FBAIN promoter generates a fusion protein when fused with the coding region of a gene to be expressed, the FBAINm promoter does not generate such a fusion protein.
  • Pex20 Pex20 terminator sequence of Yarrowia Pex20 gene (GenBank Accession No. AF054613) AscI / BsiWI 3387-2596 5' portion of Yarrowia Ura3 gene (GenBank Accession No. AJ306421) Pac I/ Sph I 6617-6095 3' portion of Yarrowia Ura3 gene (GenBank Accession No. AJ306421)
  • pDMW214 (SEQ ID NO:12; Figure 3C ) is a shuttle plasmid that replicates both in E. coli and Yarrowia lipolytica. It contained the following components: Table 5 Description Of Plasmid pDMW214 (SEQ ID NO:12) RE Sites And Nucleotides Within SEQ ID NO:12 Description Of Fragment And Chimeric Gene Components 1150-270 ColE1 plasmid origin of replication 2080-1220 Ampicillin-resistance gene (Amp R ) 2979-4256 Yarrowia autonomous replication sequence (ARS18; GenBank Accession No. A17608) Pme I/ Sph I 6501-4256 Yarrowia Leu2 gene (GenBank Accession No.
  • FBAIN::GUS::XPR comprising: • FBAIN: Yarrowia lipolytica FBAIN promoter ( PCT Publication No. WO 2005/049805 ) • GUS: E. coli gene encoding ⁇ -glucuronidase ( Jefferson, R.A. Nature, 14;342:837-838 (1989 )) • XPR: -100 bp of the 3' region of the Yarrowia Xpr gene (GenBank Accession No. M17741)
  • Construct pKUNF12T6E ( Figure 4B ; SEQ ID NO:14) was generated to integrate four chimeric genes (comprising a ⁇ 12 desaturase, a ⁇ 6 desaturase and two C 18/20 elongases) into the Ura3 loci of wild type Yarrowia strain ATCC #20362, to thereby enable production of DGLA.
  • the pKUNF12T6E plasmid contained the following components: Table 7 Description Of Plasmid pKUNF12T6E (SEQ ID NO:14) RE Sites And Nucleotides Within SEQ ID NO:14 Description Of Fragment And Chimeric Gene Components AscI/BsiWI (9420-8629) 784 bp 5' portion of Yarrowia Ura3 gene ((GenBank Accession No. AJ306421) SphI / PacI (12128-1) 516 bp 3' portion of Yarrowia Ura3 gene (GenBank Accession No.
  • FBAIN::EL1S::Pex20 comprising: • FBAIN: Yarrowia lipolytica FBAIN promoter ( PCT Publication No. WO 2005/049805 ) • EL1S: codon-optimized elongase 1 gene ( PCT Publication No. WO 2004/101753 ), derived from Mortierella alpina (GenBank Accession No. AX464731) • Pex20: Pex20 terminator sequence from Yarrowia Pex20 gene (GenBank Accession No.
  • TEF:: ⁇ 6S::Lip1 comprising: • TEF: Yarrowia lipolytica TEF promoter (GenBank Accession No. AF054508) • ⁇ 6S: codon-optimized ⁇ 6 desaturase gene ( PCT Publication No. WO 2004/101753 ), derived from Mortierella alpina (GenBank Accession No. AF465281) • Lip1: Lip1 terminator sequence from Yarrowia Lip1 gene (GenBank Accession No.
  • EL2S codon-optimized elongase gene (SEQ ID NO:15), derived from Thraustochytrium aureum ( U.S. Patent No. 6,677,145 ) • XPR: ⁇ 100 bp of the 3' region of the Yarrowia Xpr gene (GenBank Accession No. M17741)
  • GC analyses showed the presence of DGLA in the transformants containing the 4 chimeric genes of pKUNF12T6E, but not in the wild type Yarrowia control strain. Most of the selected 32 Ura - strains produced about 6% DGLA of total lipids. There were 2 strains (i.e., strains M4 and 13-8) that produced about 8% DGLA of total lipids.
  • Plasmid pDMW271 ( Figure 4C ; SEQ ID NO:17) was generated to integrate three ⁇ 5 chimeric genes into the Leu2 gene of Yarrowia strain M4.
  • Plasmid pDMW271 contained the following components, as described in Table 8: Table 8 Description Of Plasmid pDMW271 (SEQ ID NO:17) RE Sites And Nucleotides Within SEQ ID NO:17 Description Of Fragment And Chimeric Gene Components AscI/BsiWI (5520-6315) 788 bp 5' portion of Yarrowia Leu2 gene (GenBank Accession No.
  • AF260230 SphI / PacI (2820-2109) 703 bp 3' portion of Yarrowia Leu2 gene (GenBank Accession No. AF260230) SwaI / BsiWI FBAIN::MA ⁇ S::Pex20, comprising: (8960-6315) • FBAIN: Yarrowia lipolytica FBAIN promoter ( PCT Publication No. WO 2005/049805 ) • MA ⁇ 5: Mortierella alpina ⁇ 5 desaturase gene (GenBank Accession No. AFC67654) • Pex20: Pex20 terminator sequence of Yarrowia Pex20 gene (GenBank Accession No. AF054613).
  • SwaI / ClaI (8960-11055) TEF::MA ⁇ 5::Lip1, comprising: • TEF: Yarrowia lipolytica TEF promoter (GenBank Accession No. AF054508) • MA ⁇ 5: Mortierella alpina ⁇ 5 desaturase gene (GenBank Accession No. AF067654) • Lip1: Lip1 terminator sequence of Yarrowia Lip1 gene (GenBank Accession No. Z50020) PmeI / ClaI (12690-11055) Yarrowia Ura3 gene (GenBank Accession No.
  • TEF::H ⁇ 5S::Pex16 comprising: • TEF: Yarrowia lipolytica TEF promoter (GenBank Accession No. AF054508) • H ⁇ 5S: codon-optimized ⁇ 5 desaturase gene (SEQ ID NO:18), derived from Homo sapiens (GenBank Accession No. NP_037534) • Pex16: Pex16 terminator sequence of Yarrowia Pex16 gene (GenBank Accession No. U75433)
  • Plasmid pDMW271 was digested with AscI / SphI , and then used to transform strain M4 according to the General Methods. Following transformation, the cells were plated onto MMLe plates and maintained at 30°C for 2 to 3 days. The individual colonies grown on MMLe plates were picked and streaked onto MM and MMLe plates. Those colonies that could grow on MMLe plates but not on MM plates were selected as Leu2 - strains. Single colonies of Leu2 - strains were then inoculated into liquid MMLe media at 30°C and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
  • GC analyses showed the presence of ARA in pDMW271 transformants, but not in the parental M4 strain. Specifically, among the 48 selected Leu2 - transformants with pDMW271, there were 35 strains that produced less than 5% ARA of total lipids, 12 strains that produced 6-8% ARA, and 1 strain that produced about 11 % ARA of total lipids in the engineered Yarrowia. The strain that produced 11 % ARA was named "Y2047".
  • the present Example describes the construction of plasmid pZP3-P7U comprising a chimeric YAT::PsD17S::Lip1 gene, which was designed to integrate into the Pox3 locus (GenBank Accession No. XP_503244) of the Yarrowia genome. Plasmid pZP3-P7U was utilized to test functional expression of PsD17S, as described in Example 8, infra. The components of pZP3-P7U were as described in Table 9 below.
  • PsD17S codon-optimized ⁇ 17 desaturase gene (SEQ ID NO:3), derived from P. sojae
  • Lip1 Lip1 terminator sequence of Yarrowia Lip1 gene (GenBank Accession No. Z50020)
  • strain Y4070 derived from Yarrowia lipolytica ATCC #20362, capable of producing about 12% ARA relative to the total lipids via expression of a ⁇ 9 elongase/ ⁇ 8 desaturase pathway ( Figure 6A ).
  • Strain Y4070 was utilized to test the functional expression of PsD17S in Example 8 and PrD17S in
  • strain Y4070 required the construction of strain Y2224 (a FOA resistant mutant from an autonomous mutation of the Ura3 gene of wildtype Yarrowia strain ATCC #20362), strain Y4001 (producing 17% EDA with a Leu - phenotype), strain Y4001 U (producing 17% EDA with a Leu- and Ura - phenotype), strain Y4036 (producing 18% DGLA with a Leu - phenotype) and strain Y4036U (producing 18% DGLA with a Leu- and Ura - phenotype).
  • Strain Y2224 was isolated in the following manner: Yarrowia lipolytica ATCC #20362 cells from a YPD agar plate (1% yeast extract, 2% bactopeptone, 2% glucose, 2% agar) were streaked onto a MM plate (75 mg/L each of uracil and uridine, 6.7 g/L YNB with ammonia sulfate, without amino acid, and 20 g/L glucose) containing 250 mg/L 5-FOA (Zymo Research). Plates were incubated at 28°C and four of the resulting colonies were patched separately onto MM plates containing 200 mg/mL 5-FOA and MM plates lacking uracil and uridine to confirm uracil Ura3 auxotrophy.
  • Strain Y4001 was created via integration of construct pZKLeuN-29E3 ( Figure 6B ). This construct, comprising four chimeric genes (i.e., a ⁇ 12 desaturase, a C 16/18 elongase and two ⁇ 9 elongases), was integrated into the Leu2 loci of strain Y2224 to thereby enable production of EDA.
  • Plasmid pZKLeuN-29E3 (SEQ ID NO:22) RE Sites And Nucleotides Within SEQ ID NO:22 Description Of Fragment And Chimeric Gene Components BsiW I/ Asc I (7797-7002) 788 bp 3' portion of Yarrowia Leu2 gene (GenBank Accession No. AF260230) Sph VPac I (4302-3591) 703 bp 5' portion of Yarrowia Leu2 gene (GenBank Accession No.
  • Plasmid pZKLeuN-29E3 was digested with Asc I/ Sph I, and then used for transformation of Y. lipolytica strain Y2224 (i.e., ATCC #20362 Ura3 -) according to the General Methods.
  • the transformant cells were plated onto MMLeu media plates and maintained at 30 °C for 2 to 3 days. The colonies were picked and streaked onto MM and MMLeu selection plates. The colonies that could grow on MMLeu plates but not on MM plates were selected as Leu - strains. Single colonies of Leu - strains were then inoculated into liquid MMLeu at 30 °C and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
  • Strain Y4001 U was created via temporary expression of the Cre recombinase enzyme in plasmid pY116 ( Figure 6C ) within strain Y4001 to produce a Leu- and Ura - phenotype.
  • Construct pY116 contained the following components: Table 11 Description of Plasmid pY116 (SEQ ID NO:27) RE Sites And Nucleotides Within SEQ ID NO:27 Description Of Fragment And Chimeric Gene Components 1328-448 ColE1 plasmid origin of replication 2258-1398 Ampicillin-resistance gene (Amp R ) for selection in E. coli 3157-4461 Yarrowia autonomous replication sequence (ARS18; GenBank Accession No.
  • Plasmid pY116 was used for transformation of freshly grown Y4001 cells according to the General Methods.
  • the transformant cells were plated onto MMLeu + Ura plates (MMU plus Leucine) containing 280 ⁇ g/mL sulfonylurea and maintained at 30°C for 3 to 4 days.
  • Four colonies were picked, inoculated into 3 mL liquid YPD media at 30 °C and shaken at 250 rpm/min for 1 day.
  • the cultures were diluted to 1:50,000 with liquid MMLeu + Ura media, and 100 ⁇ L was plated onto new YPD plates and maintained at 30 °C for 2 days. Colonies were picked and streaked onto MMLeu and MMLeu+Ura selection plates.
  • Construct pKO2UF8289 ( Figure 7A ; SEQ ID NO:28) was generated to integrate four chimeric genes (comprising a ⁇ 12 desaturase, one ⁇ 9 elongase and two mutant ⁇ 8 desaturases) into the ⁇ 12 loci of strain Y4001U1, to thereby enable production of DGLA.
  • Construct pKO2UF8289 contained the following components: Table 12 Description of Plasmid pKO2UF8289 (SEQ ID NO:28 ) RE Sites And Nucleotides Within SEQ ID NO:28 Description Of Fragment And Chimeric Gene Components AscI / BsiWI (10304-9567) 5' portion of Yarrowia ⁇ 12 desaturase gene ( PCT Publication No. WO 2004/104167 ) EcoRI / SphI (13568-13012) 3' portion of Yarrowia ⁇ 12 desaturase gene ( PCT Publication No.
  • SwaI / BsiWI FBAINm::EgD8M::Pex20 comprising: (7055-9567) • FBAINm: Yarrowia lipolytica FBAINm promoter ( PCT Publication No. WO 2005/049805 ) • EgD8M: Synthetic ⁇ 8 desaturase mutant EgD8S-23 (SEQ ID NO:29), derived from Euglena gracilis ( U.S. Patent Application No. 11/635258 ) • Pex20: Pex20 terminator sequence from Yarrowia Pex20 gene (GenBank Accession No.
  • Swal / Pmel YAT::F.D12::OCT comprising: (7055-4581) • YAT: Yarrowia lipolytica YAT1 promoter (Patent Publication No. US 2006/0094102-A1 ) • F.D12: Fusarium moniliforme ⁇ 12 desaturase gene ( PCT Publication No. WO 2005/047485 ) • OCT terminator sequence of Yarrowia OCT gene (GenBank Accession No.
  • EXP Yarrowia lipolytica export protein
  • EXP1 Yarrowia lipolytica export protein
  • EgD8M Synthetic ⁇ 8 desaturase mutant EgD8S-23 (SEQ ID NO:29), derived from Euglena gracilis
  • Pex16 Pex16 terminator of Yarrowia Pex16 gene (GenBank Accession No.
  • PmeI / ClaI GPAT EgD9e::Lip2, comprising: (2038-1) • GPAT: Yarrowia lipolytica GPAT promoter ( PCT Publication No. WO 2006/031937 ) • EgD9e: Euglena gracilis ⁇ 9 elongase gene (SEQ ID NO:31) ( U.S. Patent Applications No. 11/601563 and 11/601564 ) • Lip2: Lip2 terminator sequence from Yarrowia Lip2 gene (GenBank Accession No.
  • the pKO2UF8289 plasmid was digested with AscI / SphI , and then used for transformation of strain Y4001U1 according to the General Methods.
  • the transformant cells were plated onto MMLeu plates and maintained at 30 °C for 2 to 3 days. The colonies were picked and streaked onto MMLeu selection plates at 30 °C for 2 days. These cells were then inoculated into liquid MMLeu at 30 °C and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by transesterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
  • GC analyses showed the presence of DGLA in the transformants containing the 4 chimeric genes of pK02UF8289, but not in the parent Y4001 U1 strain. Most of the selected 96 strains produced between 7 and 13% DGLA of total lipids. There were 6 strains (i.e., #32, #42, #60, #68, #72 and #94) that produced about 15%, 13.8%, 18.2%, 13.1%, 15.6% and 13.9% DGLA of total lipids. These six strains were designated as Y4034, Y4035, Y4036, Y4037, Y4038 and Y4039, respectively.
  • Construct pY116 ( Figure 6C ; SEQ ID NO:27) was utilized to temporarily express a Cre recombinase enzyme in strain Y4036. This released the LoxP sandwiched Ura3 gene from the genome.
  • Plasmid pY116 was used to transform strain Y4036 according to the General Methods. Following transformation, the cells were plated onto MMLeu+Ura plates (MMU plus Leucine) and maintained at 30 °C for 2 to 3 days. The individual colonies grown on MMLeu+Ura plates were picked, and streaked into YPD liquid media at 30°C and shaken at 250 rpm/min for 1 day to cure the pY116 plasmid. The grown cultures were streaked on MMLeu+Ura u plates After two days at 30 °C, the individual colonies were re-streaked on MMLeu+Ura, MMU and MMLeu plates.
  • Plasmid pZKSL-555R (SEQ ID NO:32 ) RE Sites And Nucleotides Within SEQ ID NO:32 Description Of Fragment And Chimeric Gene Components AscI / BsiWI (3321-2601) 720 bp 5' portion of Yarrowia Lys5 gene (GenBank Accession No.
  • EXP Yarrowia lipolytica export protein
  • EXP1 Yarrowia lipolytica export protein
  • EgD5S codon-optimized ⁇ 5 desaturase
  • Pex20 Pex20 terminator sequence from Yarrowia Pex20 gene (GenBank Accession No. AF054613) ClaI / PmeI YAT::RD5S::OCT, comprising: (11243-1) • YAT: Yarrowia lipolytica YAT1 promoter (Patent Publication US 2006/0094102-A1 ) • RD5S: codon-optimized ⁇ 5 desaturase (SEQ ID NO:35), derived from Peridinium sp. CCMP626 ( U.S. Provisional Application No. 60/801119 ) • OCT: OCT terminator sequence of Yarrowia OCT gene (GenBank Accession No.
  • EcoRI / PacI FBAIN::EgD5WT::Aco comprising: (9500-6716) • FBAIN: Yarrowia lipolytica FBAIN promoter ( WO 2005/049805 ) • EgD5WT: Euglena gracilis ⁇ 5 desaturase (SEQ ID NO:35; U.S. Provisional Application No. 60/801172 ) with elimination of internal Bgl II, Hind III and Nco I restriction enzyme sites • Aco: Aco terminator of Yarrowia Aco gene (GenBank Accession No. AJ001300), EcoRIClaI (9500-11243) Yarrowia Leu2 gene (GenBank Accession No. M37309)
  • the pZKSL-555R plasmid was digested with AscI / SphI , and then used for transformation of strain Y4036U according to the General Methods.
  • the transformant cells were plated onto MMLeuLys plates (MMLeu plus Lysine) and maintained at 30°C for 2 to 3 days. Single colonies were then re-streaked onto MMLeuLys plates, and then inoculated into liquid MMLeuLys at 30 °C and shaken at 250 rpm/min for 2 days.
  • the cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
  • GC analyses showed the presence of ARA in the transformants containing the 3 chimeric genes of pZKSL-555R, but not in the parent Y4036 strain. Most of the selected 96 strains produced ⁇ 10% ARA of total lipids. There were 4 strains (i.e., #57, #58, #69 and #75) that produced about 11.7%, 11.8%, 11.9% and 11.7% ARA of total lipids. These four strains were designated as Y4068, Y4069, Y4070 and Y4071, respectively. Further analyses showed that the three chimeric genes of pZKSL-555R were not integrated into the Lys5 site in the Y4068, Y4069, Y4070 and Y4071 strains.
  • strains possessed a Lys + phenotype.
  • the final genotype of strain Y4070 with respect to wildtype Yarrowia lipolytica ATCC #20362 was Ura3 -, Leu +, Lys +, GPD::F.D12::Pex20, YAT::F.D12::OCT, YAT::ME3S::Pex16, GPAT:: EgD9e::Lip2, Exp::EgD9eS::Lip1, FBAINm::EgD9eS::Lip2, FBAINm::EgD8M::Pex20, EXP::EgD8M::Pex16, FBAIN::EgD5WT::Aco, EXP::EgD5S::Pex20, YAT::RD5S::OCT.
  • Plasmid pZP3-P7U ( Figure 5 ; Example 6) was digested with AscI / SphI , and then used to transform Yarrowia lipolytica strain Y4070 according to the General Methods. Following transformation, the cells were plated onto MM plates and maintained at 30°C for 2 to 3 days. Twelve (12) transformants grown on the MM plates were picked and re-streaked onto fresh MM plates. Once grown, these strains were individually inoculated into 3 mL liquid MM at 30°C and shaken at 250 rpm/min for 2 days. The cells were collected by centrifugation, resuspended in high glucose media, and then grow for 5 days at 30°C and shaken at 250 rpm/min. The cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by transesterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
  • Fatty acids are identified as 16:0, 16:1, 18:0, 18:1 (oleic acid), LA (18:2), ALA, EDA (20:2), DGLA, ARA, ETrA (20:3), ETA and EPA; and the composition of each is presented as a % of the total fatty acids.
  • “ ⁇ 17 Activity” was calculated according to the following formula: ([EPA]/ [ARA+EPA])*100 and represents percent substrate conversion to EPA.
  • ⁇ 15 Activity was calculated according to the following formula: ([ALA]/[LA+ALA])*100 and represents percent substrate conversion to ALA.
  • JGI Joint Genome Institute
  • ORF 80222 shared 89% identity and 94% similarity with SEQ ID NO:9, with an Expectation value of O.
  • ORF48790 shared up to 40% identity and 61% similartity with SEQ ID NO:9, with an Expectation value of 6E-44. Based on these results, ORF 80222 was tentatively identified as a ⁇ 17 desaturase and was designated as "PrD17".
  • PrD17 The sequence homology of PrD17 was in turn compared with all publicly available protein sequences contained in the "nr" database (see General Methods) was also determined by conducting protein-protein BLAST searches using PrD17 (SEQ ID NO:6) as the query sequence.
  • the sequence that showed the highest degree of similarity was that of the omega-3 fatty acid desaturase of Saprolegnia diclina (GenBank Accession No. AAR20444), sharing 59% identity and 74% similarity, with an Expectation value of E-124.
  • PrD17 had 38% identity and 57% similarity with the amino acid sequence of the fatty acid desaturase of Anabaena variabilis ATCC #29413 (GenBank Accession No. ABA23809), with an Expectation value of 6E-61.
  • the codon usage of the ⁇ 17 desaturase gene of Phytophthora ramorum was optimized for expression in Yarrowia lipolytica, in a manner similar to that described in Example 2.
  • a codon-optimized ⁇ 17 desaturase gene (designated "PrD17S", SEQ ID NO:7) was designed based on the coding sequence of PrD17 (SEQ ID NOs:5 and 6), according to the Yarrowia codon usage pattern ( PCT Publication No. WO 2004/101753 ), the consensus sequence around the 'ATG' translation initiation codon, and the general rules of RNA stability ( Guhaniyogi, G. and J. Brewer, Gene, 265(1-2):11-23 (2001 )).
  • PrD17S gene was synthesized by GenScript Corporation (Piscataway, NJ) and cloned into pUC57 (GenBank Accession No. Y14837) to generate pPrD17S ( Figure 9A ; SEQ ID NO:38).
  • the present Example describes the construction of plasmid pZuFPrD17S ( Figure 9B ) comprising a chimeric FBAIN::PrD17S::Pex20 gene. Plasmid pZuFPrD17S was utilized to test functional expression of PrD17S, as described in Example 12, infra.
  • Plasmid pZuFPrD17S was constructed by using the Nco I/ Not I fragment containing the PrD17S coding region of plasmid pPrD17S to replace the Nco I/ Not I fragment of pZuF17 ( Figure 9C ; SEQ ID NO:39).
  • the components of pZuFPrD17S were as described in Table 15 below.
  • Plasmid pZuFPrD17S ( Figure 9B ; Example 11) was transformed into Yarrowia lipolytica strain Y4070 (Example 8), as described in the General Methods.
  • the transformant cells were plated onto MM selection media plates and maintained at 30°C for 2 to 3 days.
  • Ten (10) transformants grown on MM plates were picked and re-streaked onto fresh MM plates.
  • these strains were individually inoculated into 3 mL liquid MM at 30 °C and shaken at 250 rpm/min for 2 days.
  • the cells were collected by centrifugation, resuspended in high glucose media, and then grow for 5 days at 30 °C and shaken at 250 rpm/min.
  • the cells were collected by centrifugation, lipids were extracted, and fatty acid methyl esters were prepared by trans-esterification, and subsequently analyzed with a Hewlett-Packard 6890 GC.
  • Table 16 showed that there were an average of 7.5% ARA and 7.2% EPA of total lipids produced in the 10 transformants.
  • the conversion efficiency whereby PrD17S converted ARA to EPA in these 10 strains was at an average rate of about 49%.
  • the conversion efficiency was measured as described in Example 8.
  • this experimental data demonstrated that the codon-optimized ⁇ 17 desaturase gene (SEQ ID NO:5) derived from P. ramorum efficiently desaturated ARA to EPA.
  • PrD17S is identified herein as a monofunctional ⁇ 17 desaturase, catalyzing conversion of ARA to EPA.

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Claims (17)

  1. Molécule d'acide nucléique isolée qui code pour une enzyme Δ17 désaturase telle que présentée dans la SEQ ID NO:4, où ladite molécule d'acide nucléique possède des codons optimisés pour une expression dans Yarrowia relativement à la molécule d'acide nucléique telle que présentée dans la SEQ ID NO:1 ou une séquence de nucléotides isolée qui est complètement complémentaire à celle-ci.
  2. Molécule d'acide nucléique isolée selon la revendication 1, qui code pour une enzyme Δ17 désaturase telle que présentée dans la SEQ ID NO:2 ou 4, où au moins 168 codons sont des codons optimisés pour une expression dans Yarrowia ou une séquence de nucléotides isolée qui est complètement complémentaire à celle-ci.
  3. Molécule d'acide nucléique isolée selon la revendication 1, où ladite molécule d'acide nucléique isolée est une séquence telle que présentée dans la SEQ ID NO:3 ou une séquence possédant au moins 95% d'identité avec celle-ci ou une séquence de nucléotides isolée qui est complètement complémentaire à celle-ci.
  4. Molécule d'acide nucléique isolée selon la revendication 3, choisie dans le groupe constitué d'une séquence de nucléotides isolée codant pour une enzyme Δ17 désaturase telle que présentée dans la SEQ ID NO:3 et d'une séquence de nucléotides isolée qui est complètement complémentaire à celle-ci.
  5. Gène chimérique comprenant la molécule d'acide nucléique isolée selon l'une quelconque des revendications 1 à 4 liée de manière fonctionnelle à des séquences régulatrices appropriées.
  6. Espèce Yarrowia transformée comprenant la molécule d'acide nucléique isolée selon l'une quelconque des revendications 1 à 5.
  7. Espèce Yarrowia transformée selon la revendication 6, où l'espèce Yarrowia qui est transformée pour comprendre ladite molécule d'acide nucléique isolée est choisie dans le groupe constitué de Yarrowia lipolytica ATCC #20362, Yarrowia lipolytica ATCC #18944 et Yarrowia lipolytica ATCC #76982.
  8. Procédé pour la production d'acide eicosapentaénoïque comprenant:
    a) la fourniture d'une cellule hôte comprenant:
    i) une molécule nucléotidique isolée codant pour un polypeptide de Δ17 désaturase, choisie dans le groupe constitué de:
    1) une molécule nucléotidique isolée codant pour un polypeptide de Δ17 désaturase possédant au moins 95% d'identité lorsqu'il est comparé à un polypeptide présentant la séquence d'acides aminés telle que présentée dans la SEQ ID NO:2, sur la base de la méthode d'alignement Clustal W;
    2) une molécule nucléotidique isolée codant pour un polypeptide de Δ17 désaturase possédant au moins 95% d'identité lorsqu'il est comparé à un polypeptide présentant la séquence d'acides aminés telle que présentée dans la SEQ ID NO:4, sur la base de la méthode d'alignement Clustal W; et
    ii) une source d'acide arachidonique;
    b) la croissance de la cellule hôte de l'étape (a) dans des conditions dans lesquelles la molécule d'acide nucléique codant pour le polypeptide de Δ17 désaturase est exprimée et l'acide arachidonique est converti en acide eicosapentaénoïque; et
    c) éventuellement la récupération de l'acide eicosapentaénoïque de l'étape (b).
  9. Procédé pour la production d'acide eicosatétraénoïque comprenant:
    a) la fourniture d'une cellule hôte comprenant:
    i) une molécule nucléotidique isolée codant pour un polypeptide de Δ17 désaturase, choisie dans le groupe constitué de:
    1) une molécule nucléotidique isolée codant pour un polypeptide de Δ17 désaturase possédant au moins 95% d'identité lorsqu'il est comparé à un polypeptide présentant la séquence d'acides aminés telle que présentée dans la SEQ ID NO:2, sur la base de la méthode d'alignement Clustal W;
    2) une molécule nucléotidique isolée codant pour un polypeptide de Δ17 désaturase possédant au moins 95% d'identité lorsqu'il est comparé à un polypeptide présentant la séquence d'acides aminés telle que présentée dans la SEQ ID NO:4, sur la base de la méthode d'alignement Clustal W; et
    ii) une source d'acide dihomo-γ-linolénique;
    b) la croissance de la cellule hôte de l'étape (a) dans des conditions dans lesquelles la molécule d'acide nucléique codant pour le polypeptide de Δ17 désaturase est exprimée et l'acide dihomo-γ-linolénique est converti en acide eicosatétraénoïque; et
    c) éventuellement la récupération de l'acide eicosatétraénoïque de l'étape (b).
  10. Procédé pour la production d'acide α-linolénique comprenant:
    a) la fourniture d'une cellule hôte comprenant:
    i) une molécule nucléotidique isolée codant pour un polypeptide de Δ17 désaturase bifonctionnelle, choisie dans le groupe constitué de:
    1) une molécule nucléotidique isolée codant pour un polypeptide de Δ17 désaturase possédant au moins 95% d'identité lorsqu'il est comparé à un polypeptide présentant la séquence d'acides aminés telle que présentée dans la SEQ ID NO:2, sur la base de la méthode d'alignement Clustal W;
    2) une molécule nucléotidique isolée codant pour un polypeptide de Δ17 désaturase possédant au moins 95% d'identité lorsqu'il est comparé à un polypeptide présentant la séquence d'acides aminés telle que présentée dans la SEQ ID NO:4, sur la base de la méthode d'alignement Clustal W; et
    ii) une source d'acide linoléique;
    b) la croissance de la cellule hôte de l'étape (a) dans des conditions dans lesquelles la molécule d'acide nucléique codant pour le polypeptide de Δ17 désaturase bifonctionnelle est exprimée et l'acide linoléique est converti en acide α-linolénique; et
    c) éventuellement la récupération de l'acide α-linolénique de l'étape (b).
  11. Procédé selon l'une quelconque des revendications 8 à 10, dans lequel la molécule d'acide nucléique isolée code pour un polypeptide de Δ17 désaturase présentant la séquence d'acides aminés choisie dans le groupe constitué des SEQ ID NOs:2 et 4.
  12. Procédé selon la revendication 11, dans lequel la molécule d'acide nucléique isolée est telle que définie dans l'une quelconque des revendications 1 à 3.
  13. Procédé selon l'une quelconque des revendications 8 à 12, dans lequel:
    a.) la molécule d'acide nucléique isolée possède une séquence d'acide nucléique telle que présentée dans la SEQ ID NO:3, et
    b.) la cellule hôte est Yarrowia lipolytica.
  14. Procédé selon l'une quelconque des revendications 8 à 12, dans lequel la cellule hôte est choisie dans le groupe constitué: d'algues, de bactéries, de levure, d'oomycètes et de champignons, où de préférence ladite levure est une levure oléagineuse.
  15. Procédé selon la revendication 14, dans lequel la cellule hôte est un champignon choisi dans le groupe constitué de: l'espèce Thraustochytrium, l'espèce Schizochytrium et l'espèce Mortierella.
  16. Procédé selon la revendication 14, dans lequel la levure oléagineuse est choisie dans le groupe constitué de: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon et Lipomyces.
  17. Procédé selon la revendication 16, dans lequel la Yarrowia qui est transformée pour comprendre ladite molécule d'acide nucléique isolée codant pour un polypeptide de Δ17 désaturase bifonctionnelle est choisie dans le groupe constitué de: Yarrowia lipolytica ATCC #20362, Yarrowia lipolytica ATCC #18944 et Yarrowia lipolytica ATCC #76982.
EP07775772A 2006-04-20 2007-04-19 Delta-17-desaturases et leur utilisation dans la preparation d'acides gras polyinsatures Not-in-force EP2010648B1 (fr)

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CN101617040B (zh) * 2006-10-30 2012-07-04 纳幕尔杜邦公司 △17去饱和酶及其在制备多不饱和脂肪酸中的用途
US7709239B2 (en) * 2006-12-07 2010-05-04 E.I. Du Pont De Nemours And Company Mutant Δ8 desaturase genes engineered by targeted mutagenesis and their use in making polyunsaturated fatty acids
US8119860B2 (en) * 2007-04-16 2012-02-21 E. I. Du Pont De Nemours And Company Delta-9 elongases and their use in making polyunsaturated fatty acids
DK2337791T3 (da) * 2008-10-14 2013-11-04 Monsanto Technology Llc Anvendelse af fedtsyredesaturaser fra Hemiselmis SPP
WO2010066703A2 (fr) 2008-12-12 2010-06-17 Basf Plant Science Gmbh Désaturases et procédé pour la production d'acides gras polyinsaturés dans des organismes transgéniques
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JPWO2015029966A1 (ja) 2013-08-27 2017-03-02 国立大学法人京都大学 ω３不飽和脂肪酸酵素およびエイコサペンタエン酸の製造方法
US10538793B2 (en) * 2014-12-25 2020-01-21 Kyoto University ω3 fatty-acid desaturase and method for producing eicosapentaenoic acid
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CN105368851B (zh) * 2015-12-09 2019-05-07 江南大学 一种来源于寄生疫霉的ω-3脱饱和酶、含所述脱饱和酶的载体、重组微生物及其应用
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JP2009534032A (ja) 2009-09-24
CN101646766B (zh) 2013-03-27
AU2007240741B2 (en) 2013-01-31
WO2007123999A2 (fr) 2007-11-01
US7465793B2 (en) 2008-12-16
ATE542892T1 (de) 2012-02-15
DK2010648T3 (da) 2012-05-21
MX2008013306A (es) 2008-10-27
CN101646766A (zh) 2010-02-10
EP2010648A2 (fr) 2009-01-07
US20070249026A1 (en) 2007-10-25
AU2007240741A1 (en) 2007-11-01

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